Scatter labeled imaging of microvasculature in excised tissue (slime)

ABSTRACT

The present disclosure relates to a simple, fast, and low cost method for 3D microvascular imaging, termed “scatter labeled imaging of microvasculature in excised tissue” (SLIME). The method can include perfusing a contrast agent through vasculature of a tissue sample with a contrast perfusing unit (22). The contrast agent can include colloids and a dispersant. After the contrast agent is perfused through the vasculature, the vasculature of the tissue sample can be treated with a cross-linking agent delivery unit (24) providing a molecule that cross links with at least a portion of the dispersant to form a sticky, non-Newtonian polymer that prevents leakage of the contrast agent out of the vasculature of the tissue sample. The tissue sample can then be immersed in a solution comprising a clearing agent with an optical clearing unit (26) and subsequently imaged.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 62/583,645, filed Nov. 9, 2017, entitled “A POSTMORTEM 3DMICROVASCULATURE IMAGING METHOD USING OPTICAL COHERENCE TOMOGRAPHY”, theentirety of which is hereby incorporated by reference for all purposes.

TECHNICAL FIELD

The present disclosure relates generally to three-dimensional (3D)imaging of the microvasculature and, more specifically, to a simple,fast, and low cost method for 3D microvascular imaging, termed “scatterlabeled imaging of microvasculature in excised tissue” (SLIME).

BACKGROUND

Abnormal microvascular organization can be a sign of disease. In recentyears, various 3D microvascular mapping methods (e.g., micro-CT,microscopy with serial slicing, confocal microscopy) have providedvaluable information about the structure and function in various tissuesand disease states. Unfortunately, many of these methods are eithercomplex, expensive, and/or time intensive. These 3D microvascularmapping methods are not optimal for applications or studies involvingboth large volumes and large sample populations (e.g., studies involvingdiverse phenotypes, drug screening, etc.).

SUMMARY

In an aspect, the present disclosure can include a simple, fast, and lowcost method for 3D microvascular imaging, termed “scatter labeledimaging of microvasculature in excised tissue” (SLIME). SLIME is basedon optical coherence tomography (OCT), benefitting from the high speed,high resolution, wide field of view (FOV), and low cost of OCT. SLIMEbroadens the vascular imaging capability of OCT through use of an OCTcontrast agent and optical cleansing.

The method can include perfusing vasculature of a tissue sample with acontrast agent. The contrast agent can include a colloid and adispersant. After the contrast agent is perfused through thevasculature, the tissue sample can be treated with chemicals thatcross-link with at least a portion of the dispersant to form a sticky,non-Newtonian polymer that prevents leakage of the contrast agent out ofthe vasculature of the tissue sample. The tissue sample can then beimmersed in a solution comprising an optical clearing agent. After atime, in some instances, the cleared tissue sample can be imaged (e.g.,using OCT) to collect volumetric image data, then the collected imagedata can be processed and analyzed.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features of the present disclosure will becomeapparent to those skilled in the art to which the present disclosurerelates upon reading the following description with reference to theaccompanying drawings, in which:

FIG. 1 is a diagram showing a system that can be used to perform scatterlabeled imaging of microvasculature in excised tissue (SLIME) inaccordance with an aspect of the present disclosure;

FIG. 2 is a diagram showing an example configuration of thepreparation/processing unit in FIG. 1;

FIG. 3 is a process flow diagram illustrating a method for performingSLIME according to another aspect of the present disclosure;

FIG. 4 is a process flow diagram illustrating an example method forpreparing and processing a sample for the SLIME procedure in FIG. 3;

FIG. 5 includes SLIME images of 3 normal day 9 quail hearts with normalvasculature patterns;

FIG. 6 includes SLIME images of E9 quail hearts exposed to ethanolduring gastrulation with abnormal vasculature patterns;

FIG. 7 shows an example vessel density (vessel length/unit volume)calculations from an arbitrarily selected 1×1×1 mm volume from thecenter of a normal E9 quail brain;

FIG. 8 is a distance map showing the connected vessel length from nearthe beginning of the posterior cerebral artery; and

FIG. 9 shows how preliminary SLIME data includes potential abnormalitiesin an E9 quail brain after ethanol exposure.

DETAILED DESCRIPTION I. Definitions

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the present disclosure pertains.

As used herein, the singular forms “a,” “an” and “the” can also includethe plural forms, unless the context clearly indicates otherwise.

As used herein, the terms “comprises” and/or “comprising,” can specifythe presence of stated features, steps, operations, elements, and/orcomponents, but do not preclude the presence or addition of one or moreother features, steps, operations, elements, components, and/or groups.

As used herein, the term “and/or” can include any and all combinationsof one or more of the associated listed items.

As used herein, the terms “first,” “second,” etc. should not limit theelements being described by these terms. These terms are only used todistinguish one element from another. Thus, a “first” element discussedbelow could also be termed a “second” element without departing from theteachings of the present disclosure. The sequence of operations (oracts/steps) is not limited to the order presented in the claims orfigures unless specifically indicated otherwise.

As used herein, the term “imaging” can refer to the act of creating avisual representation of an interior of at least a portion of a samplefor analysis. For example, the sample can be tissue that has beenexcised or removed from a subject's body (or “excised tissue”). When theimaging is referred to as being “three-dimensional (3D)”, a volumetricvisual representation of the interior of at least the portion of thesample is created.

As used herein, the term “optical coherence tomography (OCT)” can referto an imaging technique that uses coherent light to capture micrometerresolution, two- and three-dimensional images from within a sample byoptical scattering. OCT is based on low-coherence interferometry,typically employing light with visible to near-infrared wavelengths thatpenetrates into the scattering medium.

As used herein, the term “microvasculature” can refer to the smallestvessels of the circulatory system (e.g., arterioles, venules,capillaries, or the like) that delivers blood into tissue. The vesselsof the microvasculature can average, for example, 0.3 millimeters indiameter or less. As an example, the sample can include microvasculaturewithin excised tissue.

As used herein, the term “scatter labeled imaging of microvasculature inexcised tissue (SLIME)” can refer to a convenient and cost effectivemethod for evaluating microvascular morphology and organ-levelmicrovascular organization. SLIME is operational over large fields ofview (FOVs) in large cohort studies in a variety of excised tissuesamples from a variety of subjects. SLIME generally includes the stepsof tissue preparation and processing (e.g., delivering a contrast agent,cross-linking the contrast agent to prevent leakage, and opticalclearing the sample), imaging, and data processing.

As used herein, the term “contrast agent” can refer to a substance usedto increase the visibility of a structure (e.g., blood vessels,gastrointestinal tissue, lymphatic tissue, etc.) within the sample beingimaged. Contrast agents can be classified based on the imagingmodalities in which they are being used. The contrast agent can beformed of a colloid and a dispersant.

As used herein, the term “colloid” can refer to a mixture in which onesubstance of microscopically dispersed insoluble particles is suspendedthroughout another substance. For example, the insoluble particles caninclude TiO₂ and the other substance can be a water-based solution.

As used herein, the term “dispersant” can refer to a surfactant polymeror other substance added to a colloid suspension to improve theseparation of particles and to prevent settling or clumping. As anexample, the dispersant can be polyvinyl alcohol/acetate (PVA).

As used herein, the term “slime” can refer to a sticky, non-Newtonianpolymer formed by contacting a cross-linking solution (e.g., containingboric acid) with a dispersant.

As used herein, the terms “cross-linking” and “crosslinking” can referto the act of creating a chemical bond (e.g., covalent or ionic) thatlinks one polymer chain to another. Cross-linking can change thematerial properties of the linked polymer molecules.

As used herein, the terms “optical clearing” and “clearing” can refer tothe act of minimizing light scatter from a sample by exposing the sampleto a clearing agent. For example, the sample can be immersed in amodified CUBIC-I clearing agent containing boric acid to clear thesample for imaging.

As used herein, the term “subject” can be used interchangeably and referto any warm-blooded organism including, but not limited to, a humanbeing, a pig, a rat, a mouse, a bird, a dog, a cat, a goat, a sheep, ahorse, a monkey, an ape, a rabbit, a cow, etc.

II. Overview

The present disclosure can include a simple, fast, and low cost methodfor 3D microvascular imaging, termed “scatter labeled imaging ofmicrovasculature in excised tissue” (SLIME). SLIME is an integratedmethod with tissue processing, imaging and data processing. SLIME isboth a convenient and cost effective method for evaluating microvascularmorphology over a large FOV in large cohort studies. However, SLIME canbe applied to any biological vessels or lumens that can be perfused(e.g., lymphatic tract, digestive tract, etc.).

As an example, SLIME can be used with optical coherence tomography (OCT)(however, other imaging modalities can be used with the only changebeing a different colloidal-based contrast agent). As such, thevasculature can be filled with a specialized OCT contrast agent toproduce a strong OCT signal. The contrast agent is rapidly cross-linkedto prevent leakage. The tissue is optically cleared to reduce lightscattering and increase the accessible imaging depth. Because thecontrast agent is not affected by optical clearing, the combined effectgenerates high OCT contrast between the filled vasculature and the restof the tissue. Effective 3D image processing specific for SLIME allowsvisualization and quantification of microvascular morphology. Theoverall SLIME pipeline allows users to obtain 3D microvascular maps ofsmall animal organs with minimal operation time.

III. Systems

One aspect of the present disclosure can include a system 10 (FIG. 1)that can be used to perform scatter labeled imaging of microvasculaturein excised tissue (SLIME). The imaging modality used in connection withSLIME can be optical coherence tomography (OCT), but other imagingmodalities can be utilized with only minimal changes to the SLIMEprocedure. SLIME provides a convenient and cost effective way toevaluate properties (e.g., morphology, 3D organization, etc.) of acomponent within an excised tissue sample. The component can include anybiological vessels or lumens that can be perfused (e.g., lymphatictubes, blood vessels (e.g., micro vessels), the digestive tract, and thelike). SLIME can provide high-resolution images with a wide field ofview (FOV) and imaging depth at a high speed without suffering fromshadowing artifacts.

The system 10 includes a preparation/processing unit 12, an imaging unit14, and an image processing unit 16. One or more of thepreparation/processing unit 12, the imaging unit 14, or the imageprocessing unit 16 can perform computer-aided actions, which are storedin one or more non-transitory memory units and executed by one or moreprocessors. In some instances, each of the preparation/processing unit12, the imaging unit 14, and the image processing unit 16 can be uniqueentitles (e.g., located at different places). However, in otherinstances, functionalities of one or more of the preparation/processingunit 12, the imaging unit 14, and the image processing unit 16 can becombined.

The preparation/processing unit 12 can broaden the imaging capability ofOCT and other imaging modalities by combining the use of a contrastagent with optical clearing. FIG. 2 shows an examplepreparation/processing unit 12, including a contrast perfusion unit 22,a cross-linking agent delivery unit 24, and an optical clearing unit 26that work together to prepare the sample for imaging by the imaging unit14.

The contrast perfusion unit 22 can fill the component of the tissuesample with a contrast agent. The tissue sample can be excised or can bein a natural state inside the subject's body pre-excision. The contrastagent maintains a low viscosity necessary for perfusion of the contrastagent through the component of the tissue sample. As an example, thecontrast agent can include a colloid and a dispersant. For example, thecolloid can be chosen to provide a strong signal based on the imagingmodality being used (e.g., when the imaging modality is OCT, the colloidand dispersant can be chosen to produce a strong OCT signal) and thedispersant can be chosen to maintain the low viscosity. As an example,when the imaging modality is OCT, the colloid can include TiO₂nanoparticles in a water-based solution (which can optimizebackscattering), while the dispersant can include low molecular weight(e.g., Mw ˜10,000) polyvinyl alcohol/acetate (PVA) (which can optimizeviscosity). In some instances, the contrast agent can include apreservative for longer preservation, like paraformaldehyde (PFA).

While perfusion with the contrast agent requires low viscosity, keepingthe contrast agent within the component of the tissue sample requireslow fluidity. Accordingly, after perfusion with the contrast agent, thetissue sample can be treated with a cross-linking agent to lower thefluidity. The tissue sample can be excised or can be in a natural stateinside the subject's body pre-excision. Accordingly, thepreparation/processing unit 12 can also include a cross-linking agentdelivery unit 24 to deliver the cross-linking agent to the component ofthe tissue sample.

At least a portion of the cross-linking agent delivered by thecross-linking agent delivery unit 24 can rapidly cross-link to a portionof the binding agent of the contrast agent. The cross-linking agent caninclude borate ions, which can cross-link with the dispersant of thecontrast agent within a few seconds to form a sticky, non-Newtonianpolymer that clogs any openings in the component of the tissue sample,such as those openings that may have been created during the excisionprocedure, to prevent contrast agent leakage.

Additionally, the preparation/processing unit can include an opticalclearing unit 26, which can perform optical clearing on an excisedtissue sample for a time that varies based on a size of the excisedtissue sample. The optical clearing unit 26 can aid in the clearing witha mechanical force, like gentle shaking. To perform the opticalclearing, the excised tissue sample is immersed in a clearing agent(e.g., CUBIC-1 for OCT imaging to reduce light scattering and increasethe accessible imaging depth, but different clearing agents can be used)that has been modified to contain boric acid. Optical clearing is simpleand fast, requiring only the immersion of the excised tissue sample inthe clearing agent that has bene modified to contain boric acid. Theclearing agent can generate sufficient transparency for imaging, whileborate ions of the boric acid further stabilize the contrast agentinside the excused tissue sample. Because the contrast agent is notaffected by the optical clearing, the combined effect generates highcontrast between the filled component and the rest of the tissue sample.

Referring again to FIG. 1, the imaging unit 14 can record raw datacorresponding to one or more images of the excised tissue sample afterthe excised tissue sample emerges from the preparation/processing unit.As an example, the imaging unit 14 can record the one or more images ofthe excised tissue sample while immersed within the clearing agentmodified with boric acid. In some instances, the imaging unit 14 canrecord one or more OCT images of the excised tissue sample. However,other imaging modalities can be utilized by the imaging unit 14.

The image processing unit 16 can receive the raw data from the imagingunit 14 and process the raw data to provide information about the imagesand/or the images themselves. The raw data can provide high contrastimages of the component of the excised tissue sample. For example, theimage processing unit 16 can employ one or more image processing tools,such as ImageJ, Almira, or the like, to provide visualization and/orquantitate information about the one or more images that can be used forstatistical analysis.

As an example, the image processing unit 16 can perform the followingprocessing on OCT raw data. A multi-iterative 3D median filter can beapplied to the raw data to reduce speckle noise. Coherence enhancingdiffusion filtering can be applied to reduce noise and improvecontinuity of the component of the tissue sample. Image contrast andintensity uniformity can be further improved by background subtractionand 3D unsharp filtering. In addition to qualitative visualinterpretation, processed data can be segmented through simple intensitythresholding and skeletonized into 3D graphical data for quantitativeanalysis. Topological assessments (e.g., skeletonization) can be readilyperformed with existing software tools.

IV. Methods

Another aspect of the present disclosure can include methods 30 and 40(FIGS. 3 and 4) for performing scatter labeled imaging ofmicrovasculature in excised tissue (SLIME). SLIME provides a convenientand cost effective way to evaluate properties (e.g., morphology, 3Dorganization, etc.) of a component within an excised tissue sample. Thecomponent can include any biological vessels or lumens that can beperfused (e.g., lymphatic tubes, blood vessels (e.g., micro vessels),the digestive tract, and the like). SLIME can provide high-resolutionimages with a wide field of view (FOV) and imaging depth at a high speedwithout suffering from shadowing artifacts. The methods 30 and 40 can beexecuted using at least a portion of the system 10 shown in FIGS. 1 and2 and described above.

Referring now to FIG. 3, illustrated is a process flow diagram of amethod 30 for performing SLIME according to another aspect of thepresent disclosure. As noted, the imaging modality used in connectionwith SLIME can be optical coherence tomography (OCT), but other imagingmodalities can be utilized with only minimal changes to the SLIMEprocedure. At step 32, a sample can be prepared and processed to broadenthe imaging capability of OCT and other imaging modalities by combiningthe use of a contrast agent with optical clearing. The preparation andprocessing of the sample can include the steps of the method 40illustrated in FIG. 4.

At step 42, a component of the tissue sample can be perfused with acontrast agent to fill the component of the tissue sample with thecontrast agent. The tissue sample can be excised or can be in a naturalstate inside the subject's body pre-excision. The contrast agentmaintains a low viscosity necessary for perfusion of the contrast agentthrough the component of the tissue sample. As an example, the contrastagent can include a colloid and a dispersant. For example, the colloidcan be chosen to provide a strong signal based on the imaging modalitybeing used (e.g., when the imaging modality is OCT, the colloid anddispersant can be chosen so that the component produces a strong OCTsignal) and the dispersant can be chosen to maintain the low viscosity.As an example, when the imaging modality is OCT, the colloid can includeTiO₂ nanoparticles in a water-based solution (which can optimizebackscattering), while the dispersant can include low molecular weight(e.g., Mw ˜10,000) polyvinyl alcohol/acetate (PVA) (which can optimizeviscosity). Different colloids and/or dispersants can be chosen fordifferent imaging modalities. In some instances, the contrast agent caninclude a preservative for longer preservation, like paraformaldehyde(PFA).

After perfusion with the contrast agent, at step 44, the component ofthe tissue sample (either excised or in a natural state) can be treatedwith a cross-linking agent to ensure low fluidity to keep the contrastagent within the component of the tissue sample. The cross-linking agentcan rapidly cross-link with at least a portion of the dispersant to forma sticky, non-Newtonian polymer that closes any openings in thecomponent of the tissue sample and prevents leakage of the contrastagent out of the component of the tissue sample.

At step 46, the tissue sample can be immersed in a solution comprising aclearing agent. The clearing agent can include a traditional clearingagent (CUBIC-1 for OCT imaging) that has been modified to contain boricacid. Optical clearing is simple and fast, requiring only the immersionof the excised tissue sample in the clearing agent that has benemodified to contain boric acid. The clearing agent can generatesufficient transparency for imaging, while borate ions of the boric acidfurther stabilize the contrast agent inside the excused tissue sample.Because the contrast agent is not affected by the optical clearing, thecombined effect generates high contrast between the filled component andthe rest of the tissue sample.

Referring again to FIG. 3, at step 34, the prepared and processed samplecan be imaged. When the sample is imaged, raw data corresponding to oneor more images of the sample immersed in the clearing agent solution canbe recorded. At step 36, raw data related to the imaging of the samplecan be processed. The processing can enable assessing whether a diseasestate exists in the sample and/or quantifying aspects of morphology ofthe component of the tissue sample based on analyzing the images of thetissue sample. The analysis can be performed according to an imageprocessing procedure.

An example when the imaging unit records OCT raw data, the processingcan include one or more of the following. A multi-iterative 3D medianfilter can be applied to the raw data to effectively reduce specklenoise. Coherence enhancing diffusion filtering can be applied to reducenoise and improve continuity of the component of the tissue sample.Image contrast and intensity uniformity can be further improved bybackground subtraction and 3D unsharp filtering. In addition toqualitative visual interpretation, processed data can be segmentedthrough simple intensity thresholding and skeletonized into 3D graphicaldata for quantitative analysis. Topological assessments (e.g.,skeletonization) can be readily performed with existing software tools.

V. Experimental

The following experiment shows a simple, fast, low costthree-dimensional (3D) microvascular mapping method, which uses opticalcoherence tomography (OCT), and is referred to as “scatter labeledimaging of microvasculature in excised tissue” (SLIME). SLIME combinesoptical clearing with a scatter-based OCT contrast agent, greatlyextending the OCT imaging depth in excised tissues, while maintaining asimple and lost cost approach that does not require extensive knowledgeof OCT. The following experimental results are shown for the purpose ofillustration only and are not intended to limit the scope of theappended claims.

Method Steps of SLIME

First, the SLIME contrast agent was formulated to optimize both itsbackscattering and viscosity parameters. While perfusion requires lowvelocity, keeping the contrast agent in the blood vessel requires lowfluidity. After perfusion, treat tissue with a sticky non-Newtonianpolymer (referred to as the toy “Slime”) to clog any vessel openings andeffectively prevent contrast agent leaking. After clogging any vesselopenings, the tissue/organ is harvested and immersed in an opticalclearing solution. The optical clearing solution generates sufficienttransparency for OCT imaging. Additionally, the optical clearingsolution can further stabilize the contrast agent in the tissue/organ.After clearing, the sample can be imaged directly in the clearingsolution with conventional OCT. The microvasculature organization can bereflected through intensity contrast directly in a single scan.

SLIME Contrast Agent

The contrast agent stock solution was a mixture of 12.5% w/w titaniumdioxide (TiO₂) nanoparticles, 12.5% w/w polyvinyl alcohol/acetate (PVA),0.5% w/w glycerol and 2.5% w/w commercial paint wetting agent in water.The concentration of each ingredient can be adjusted to achievedifferent image contrast and solution viscosity. To prevent aggregationof TiO₂ nanoparticles in PVA solution, concentrated TiO₂ water colloidand PVA solution were prepared separately and mixed to make the finalcolloid solution just before use. A commercial water-based paint wettingagent (Solsperse 27000, Lubrizol) is diluted before adding TiO₂nanoparticles (Aerosil Aeroxide P25, Evonik, anatase, primary particlesize =21 nm) to make a 25% w/w colloid solution. For each gram of TiO₂nanoparticles, 200 mg of wetting agent was used. After gentle stirringusing a magnetic stirrer, the mixture is sonicated using a 250 W, 20kHz, ultrasound homogenizer. Sonication at full power for 1 minute issufficient to disperse 25 g of TiO₂ nanoparticles in 5 g of wettingagent dissolved in 70 g of water. Low molecular weight PVA (M_(w)9000-10000, Sigma, 80% hydrolyzed) was dissolved in deionized water at80° C. under constant stirring to make a 25% w/w solution. The 25% TiO₂colloid is mixed 1:1 with the 25% PVA solution at 80C. 0.5 w/w glycerolis added to the mixture as a plasticizer. The mixture was furthersonicated for 1 min per 100 g of mixture. The final colloidal solutionshould be very stable and have a shelf life of more than 3 months. Ifsmall amounts of precipitation occur during storage, sonication can beused to re-disperse the constituents. The stock solution was diluted 4:1in 5× phosphate buffered saline (PBS) or 20% paraformaldehyde (PFA) tomake a working solution.

Crosslinking Solution

4% w/v boric acid was used as the crosslinking solution. The pH of thesolution was titrated to 7.8 with 1 N sodium hydroxide. The solution wasstored at 37° C. Precipitation gradually occurs when the temperature islower than the storage temperature, but will not significantly affectthe performance of crosslinking. 0.05% w/v NaN₃ may be added as amicrobicide for longer storage time.

Clearing Solution

The clearing solution was modified from the Scale CUBIC-1 clearingagent, which contains 25% w/w urea, 25% w/w Quadrol, 15% w/wTriton-X100, 15% w/w crosslinking solution and 15% w/w water. 25% w/wglycerol can be used as an alternative to Quadrol. To prepare 100 gclearing solution, 25 g Quadrol and 15 g crosslinking solution weredissolved in 15 mL DI water with continuous stirring using a magneticstirrer hotplate. Heating or 20 kHz sonication can be applied toaccelerate the solubilization, but the temperature of the solutionshould be maintained below ˜50° C. After the solution was completelymixed, 25 g of urea was added and dissolved by continuous stirring. Thesolution was cooled to room temperature and weighed. To compensate forthe evaporation during the previous steps, DI water is added to make thetotal mass of the solution 85 g. Then 15 g of Triton X-100 is added andstirred slowly at room temperature until completely mixed. The solutioncan be stored at room temperature for up to 3 months.

Perfusion

Prior to perfusion, a working solution was made by mixing the SLIMEcontrast agent stock solution 4:1 with 5× PBS or 20% PFA. The mixturewas gently shaken for 5 minutes with a nutating mixer. Vigorous shakingshould be avoided to prevent air bubble formation. In general, theperfusion protocol is identical to generic perfusion fixation protocolsexcept the fixative solution is replaced with the SLIME contrast agent.Several small modifications to the SLIME perfusion protocol are neededto accommodate different animal models and tissues of interest. Forembryos from smaller animals (e.g., mouse, avian), the animal can beperfused directly with the working solution. For larger animals (e.g.,adult mouse and rat), the vasculature should be first flushed withanticoagulation solutions such as heparinized saline. The left ventricleis a convenient cannulation site for SLIME perfusion. Alternatively, ifintact coronary circulation is being studied, perfusion can be done atthe aortic arch or abdominal aorta. Because the contrast agent is white,the endpoint of perfusion is easily observed at low magnification. Thetissues of interest turn pale and the large vessels become distinctlywhite. After perfusion, the tissues of interest are rinsed using theborate crosslinking solution. For tissues buried under other structures(e.g., brain under the skull), the crosslinking solution can be injectedinto the interfaces and cavities near the tissue using a syringe. Aftera brief waiting period of about 30 s, the tissues can be dissected outof the body using appropriate tools and procedures. As the tissues aresurrounded by the crosslinking agent, the contrast agent rapidly turninto a viscous and sticky material, which occludes vessels once thedissection takes place. If the vessels are pressurized or crosslinkingis insufficient, leaking of contrast agent may occur. Adding morecrosslinking agent with gentle compression can effectively stop theleakage. If contrast agent collects on the sample surface and impedessample handling (e.g., sticks to the instruments), the contrast agent onthe sample surface can be hardened and peeled off with a brief rinse offormamide

Optical Clearing

After dissection, samples were cleared by immersing in excess modifiedScale CUBIC-I solution. Clearing time depends on the size of the samplewith bigger samples taking significantly longer to clear. For instance,a stage 36 quail embryo heart (˜2.5×4×4 mm) only requires about 12 hoursto clear. The brain from the same stage quail embryo (˜3.5×8×8 mm)requires more than 24 hours to clear. It can take more than 5 days toclear the brain of a P5 mouse (˜6×12×12 mm). To reduce the time forclearing large tissue samples, the samples can be dissected into ˜2 mmthin slices using a vibratome, which reduces the clearing time to lessthan 24 hours. For faster clearing speed, the clearing solution can bereplaced with fresh solution every 2 hours.

OCT Imaging

The cleared SLIME sample can be directly imaged using conventional OCT.The sample was immersed in optical clearing solution during OCT imaging.To eliminate reflection from the curved liquid-air interface, amicroscope coverslip was placed on top of the sample. Because the lightwas polarized, signal from the cleared tissue can be suppressed bycross-polarizing the light between the sample arm and the reference arm.In contrast, TiO₂ nanoparticle clusters depolarize the light, andtherefore are not strongly affected by the cross-polarization. Since theintensity ratio between the contrast agent and the cleared tissue washigh with this simple setup, sufficient image contrast can be obtainedwith just a single volume scan. Conventional OCT with Gaussian focusingonly has the optimal lateral resolution in the Rayleigh range. As focalpower increases, resolution increases and depth of focus decreases. Withconventional OCT, both large axial range and high resolution can beachieved through volume stitching. In this case, multiple volume scansshould be taken by focusing the beam at different depths of the tissue.Alternatively, OCT with Bessel beam illumination could be used withSLIME to achieve both high resolution and extended depth in singlescans.

Data Processing and Analysis

Because SLIME raw data provides high contrast images of blood vessels,simple automated image processing can be performed to compute usefulinformation using tools that are readily available. For example, amulti-iterative 3D median filter can be applied to reduce speckle noise.Coherence enhancing diffusion filtering can be applied to further reducenoise and improve vessel continuity. Image contrast and intensityuniformity can be further improved by background subtraction and 3Dunsharp filtering. In addition to qualitative visual interpretation,processed vascular data can be segmented through simple intensitythresholding and skeletonized into 3D graph data for quantitativeanalysis. Topological assessments (e.g., skeletonization) can be readilyperformed with existing tools (e.g., Amira).

In this case, data was processed using customized code and imageprocessing software. Conventional OCT processing (e.g., wavenumberlinearization, Fourier transform) was performed on SLIME raw data tomake typical OCT structure images. For higher image contrast, logcompression can be omitted. If necessary, regions of interest aremanually segmented to minimize processing time before further imageprocessing. Some quantitative data analysis, such as cross-sectionalarea and circumference of large vessels can be performed directlywithout further image processing. To perform vessel cross-sectional areaor circumference calculations, orthogonal slices to a vessel of interestare selected and the area or circumference are manually segmented andcomputed with Fiji (ImageJ).

Additional processing of the SLIME images was performed to reduce noiseand improve data interpretability after log compression. First, 3iterations of a 3D median filter were applied to reduce noise. Next, thedata were filtered with a 3D coherence-enhancing diffusion filter toremove gaps in the vessel caused by OCT speckle noise. These filterswere anisotropic and smooth along the length of linear structures (e.g.,vessels) which effectively reduces speckle noise and enhances vesselconnectivity, which improves the ease of visual interpretation andautomated vessel segmentation. Occasionally, samples were notsufficiently cleared and background subtraction is used. In thissituation, background is estimated with a down-sampled, blurred image.To further improve vessel contrast, the data are processed using eitheran unsharp mask or high pass filter.

In this work, data skeletonization was performed with Amira (ThermoFisher Scientific). One of the most frequently used visualizationmethods demonstrated in this manuscript is referred to as “volumerendering”—a ray tracing based rendering technique that simulates thevoxels of 3D objects as solid matter. Different voxel values are mappedto different optical properties. Usually larger voxel values aresimulated as more solid and scattering materials, while smaller valuesare more transparent. A simulated light source casts rays onto theobjects, creating reflections, diffusions and shadows to form an image.Volume rendering is most suitable for studying vessel morphology nearthe surface. For visualizing internal structures deep in the volume, thesample can be cropped/clipped into smaller/thinner volumes.

To get a binary map of the vasculature for skeletonization, simpleintensity-based thresholding was used. With skeletonized data, locallengths, locations and orientations of local vessel segments can beevaluated and used to calculate other parameters such as vessel lengthdensity and local tortuosity. Here, vessel length density is defined andcalculated by counting the total length of the vessel in a unit volumeof tissue. Because vessel diameter can vary significant due topreparation and measurement errors, we believe vessel length density isa better measurement of vessel density compared to volume fraction.

Animals

According to IACUC guidelines at Case Western Reserve University, thepolicy on the use of Avian Embryos states that, “If embryos will besacrificed prior to 3 days before hatching, the research will not besubject to IACUC review.” Domestic quail embryos typically hatch aroundembryonic day 17. Therefore, IACUC approval was not required for quailembryos in this study. Fresh fertilized quail eggs were purchased from acommercial supplier. The eggs were incubated in an egg incubator at 37°C. with near saturated humidity, until they achieved the desireddevelopmental stages.

For the preliminary tests on rodents, all procedures were conducted inaccordance with Case Western Reserve University IACUC-approvedprotocols. All rodents were leftover wild type animals from unrelatedstudies. The rodents were euthanized by perfusion fixation underanesthesia prior to SLIME perfusion.

Quail Fetal Alcohol Syndrome (FAS) Model

The primary reason to use the quail FAS animal model was to helpvalidate the technique. The FAS model was easy to use and causes a largevariety of cardiovascular structural abnormalities at different scalelevels. It was hypothesized that some abnormalities could also occur atthe microvascular level, and would be diverse in their presentation. Thegoal of the work was to demonstrate SLIME' s ability to identifymicrovascular abnormalities that may be missed using traditionaltechniques.

To generate quail embryos with FAS, a well-established protocol wasused. In brief, quail eggs were incubated at 37° C. until gastrulation(˜21 hours). The eggs were then injected with 40 μl of 50% ethanol innormal saline solution. The injection sites were sealed, and the eggswere kept in the incubator until they reached the desired developmentalstages.

To validate SLIME's ability to capture microvascular phenotypes acrossan entire organ, the SLIME results of 6 normal quail embryo hearts and 6quail embryo hearts from the FAS model were compared. The coronarypatterns from the SLIME data were evaluated by a human expert.

To demonstrate SLIME's ability to make quantitative measurements, somestage 36 quail embryo brains from both groups were examined Because thebrains have large volume, diverse vascular morphology and highstructural symmetry, they were ideal for validating and demonstratingquantitative assessment. Vessel length density, length to specificpoints and local tortuosity measurements were performed. Using thesymmetry of the brain, the measurements were validated by comparinganalysis made on both sides of the brain.

Results

SLIME is a convenient and cost effective method for evaluatingmicrovascular morphology over a large FOV in large cohort studies in avariety of animal tissues (including those of rodents). SLIME broadensthe vascular imaging capability of OCT and provides the benefits of highspeed, high resolution, and a wide FOV. Even in a slow OCT system with10 kHz line rate, it takes less than 2 minutes to acquire a 4×4×3 FOV(XYZ) at 4×4×6 μm voxel sampling, which is sufficient to resolve theorganization of the microvasculature in most tissues tested. Further,SLIME does not have shadowing artifacts that are common in OCT and theimaging depth is much greater and only limited by the optics of the OCTsystem (˜2 mm/single scan for capillary resolution).

In some disease models, microvascular phenotypes at the organ level canvary significantly between individuals. Investigating a small number ofsamples and small portions of a sample can be inconclusive ormisleading. However, with traditional imaging techniques (e.g., confocalmicroscopy), it is often resource intensive to acquire high resolution,large FOV images for a large population. This problem can be effectivelyaddressed with SLIME, which is capable of mapping the microvasculatureat the organ level with micrometers resolution and millimeters FOVwithin minutes. As a demonstration, SLIME was used to producehigh-resolution data of quail embryo hearts in a well-established modelof fetal alcohol syndrome (FAS) with diverse cardiovascular defects(FIGS. 5 sf 6). Various atypical microvascular phenotypes (e.g., chaoticalignment, reduced vessel density, abnormal organization) wereidentified. These microvascular abnormalities can be subclinical, butmay eventually lead to more serious heart defects.

Quantifying aspects of vascular morphology across an entire organ cangenerate numerous insights about blood perfusion and metabolism in bothnormal and disease states. Because of the high contrast of SLIME imagesand the ability to skeletonize the data, various quantitativemeasurements can be easily made. As a proof of concept, thequantification of SLIME images were demonstrated for a stage 36 quailembryo brain. Vessel length density (total vessel length/tissue volume(FIG. 7) and distance maps indicating vessel length to specific pointsof interest can be readily calculated from SLIME data using existingtools (FIG. 8). Further, quantitative measurements of SLIME images canbe used as metrics to assess abnormalities. Normal embryonic quailbrains show symmetrical vascular patterning between the left and rightside. In the brains of the quail FAS model, SLIME data showed cleardifferences in local tortuosity between the left and right optical lobes(FIG. 9).

From the above description, those skilled in the art will perceiveimprovements, changes and modifications. Such improvements, changes andmodifications are within the skill of one in the art and are intended tobe covered by the appended claims.

The following is claimed:
 1. A method comprising: perfusing a contrast agent through vasculature of a tissue sample, wherein the contrast agent comprises a colloid and a dispersant; treating the vasculature of the tissue sample with a molecule that cross links with at least a portion of the dispersant to form a sticky, non-Newtonian polymer that prevents leakage of the contrast agent out of the vasculature of the tissue sample; and immersing the tissue sample in a solution comprising a clearing agent.
 2. The method of claim 1, further comprising recording one or more images of the tissue sample in the solution comprising the clearing agent.
 3. The method of claim 2, wherein the one or more images are optical coherence tomography (OCT) images and the contrast agent is an OCT contrast agent.
 4. The method of claim 3, wherein the colloid comprises metal nanoparticles.
 5. The method of claim 4, wherein the metal nanoparticles comprises TiO₂.
 6. The method of claim 1, wherein the dispersant comprises poly vinyl alcohol/acetate (PVA) and the molecule that cross-links with the portion of the dispersant cross-links with the PVA.
 7. The method of claim 6, wherein the molecule that cross-links with the PVA is boric acid.
 8. The method of claim 7, wherein the solution comprising the clearing agent further comprises boric acid to further prevent leakage of the contrast agent from the tissue sample.
 9. The method of claim 1, wherein the vasculature comprises perfusable connected vessels or lumen in the tissue sample.
 10. The method of claim 9, wherein the tissue sample comprises blood vessels, lymph tubes, or digestive tract.
 11. The method of claim 1, wherein the vasculature of the tissue sample exhibits a high contrast with the tissue sample in the one or more images.
 12. The method of claim 1, further comprising performing an image processing procedure on the one or more images, wherein the one or more images are optical coherence tomography (OCT) images.
 13. The method of claim 12, wherein the image processing procedure comprises one or more of: applying a multi-iterative three-dimensional (3D) median filter to data related to the one or more OCT images to reduce speckle noise; applying coherence-enhanced diffusion filtering to reduce noise and improve vessel continuity in the data related to the one or more OCT images; improving image contrast and intensity uniformity by background subtraction and/or 3D unsharp filtering of the data related to the one or more OCT images; and segmenting the data related to the one or more OCT images through intensity thresholding and skeletonizing the segmented data related to the one or more OCT images into 3D graph data for quantitative analysis.
 14. The method of claim 1, wherein the tissue sample is affected by the clearing agent and the contrast agent is not affected by the clearing agent, thus enhancing contrast between the vasculature of the tissue sample and the tissue sample.
 15. The method of claim 1, further comprising assessing whether a disease state exists in the tissue sample based on analyzing the one or more images of the tissue sample.
 16. The method of claim 1, wherein the analyzing further comprises quantifying aspects of morphology of the vasculature in the tissue sample. 